recording of mesenteric afferent nerve activity in an ex vivo mouse jejunum segment

Recording of Mesenteric Afferent Nerve Activity in an Ex Vivo Mouse Jejunum Segment

In this procedure, using a micromanipulator, lower the tip of the suction electrode into the organ bath. Then, gently aspirate some Krebs solution from the organ bath so that the tip of the electrode is submerged in the Krebs solution. Ensure that the Krebs solution covers the wire electrode inside the suction electrode.
Next, position the tip of the suction electrode immediately next to the transected afferent nerve strand, and draw the transected nerve strand into the capillary over its entire length. Maneuver the tip of the electrode towards the adipose tissue, and aspirate it into the glass capillary with the plunger, thereby mechanically sealing the nerve in the capillary from the contents of the organ bath.
To verify the recording of afferent nerve activity, perform a ramp distension-induced increase in afferent firing by closing the output port to lead to a gradual rise in pressure in the intestinal segment. Only perform the desired experimental protocol when three consecutive ramp distensions yield a reproducible multi-unit discharge.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of the jejunal afferent nerve
<ol>
	<li>Perform rodent euthanasia of the adolescent or adult rodent that has been approved prior to the experiment by the local Ethical Committee (e.g., terminal sedation followed by cardiac puncture, cervical dislocation, etc.). 
	NOTE: We used cervical dislocation to sacrifice the animals, thus resulting in experiments without the need for further anesthesia or post-surgical care as tissues are further processed in vitro. 
	NOTE: Age has been shown to attenuate mesenteric afferent mechanosensory functions, we therefore advise researchers to adhere to a specific age group for the duration of a single experiment.</li>
	<li>Place the sacrificed laboratory animal in the supine position and perform an abdominal midline incision through the skin and abdominal muscle layer using a scalpel, extending from the xiphoid process until the pubic bone.</li>
	<li>Bathe the abdominal cavity with cold Krebs solution in order to prevent the intra-abdominal tissues from drying out (Krebs composition: 120.03 mM NaCl, 6.22 mM KCl, 1.57 mM NaH2PO4, 15.43 mM NaHCO3, 1.21 mM MgSO4, 11.52 mM D-glucose and 1.52 mM CaCl2).</li>
	<li>Rapidly excise the entire jejunum using sharp scissors by excising approximately 20 cm of the small bowel starting immediately distally of the duodenojejunal flexure, taking care not to damage surrounding structures and keeping the bowel&#39;s mesentery, which contains jejunal blood vessels and afferent nerves, intact. 
	NOTE: For the mere jejunal dissection in the abdominal cavity, one does not need to use a stereomicroscope, as this can be easily visualized with the naked eye.</li>
	<li>Place the excised jejunum in an ice-cold Krebs solution and keep it on ice while continuously oxygenating the Krebs solution with carbogen (95% O2, 5% CO2).</li>
	<li>Cut the jejunum with sharp scissors in approximately 3 cm long loops. Observe the mesenteric bundle containing the vessels and splanchnic nerve somewhere near the center of the respective loop.</li>
	<li>Flush each segment with Krebs solution using a blunt catheter to remove luminal contents and chyme, as these contain digestive enzymes that will accelerate the deterioration of the tissue sample in vitro. 
	NOTE: Take care not to damage the lumen of the loop during flushing, as the destruction of the villi will result in the release of mediators that can alter the afferent nerve activity.</li>
	<li>Identify the segment to measure the afferent nerve activity (e.g., the first jejunal segment distally of the ligament of Treitz or the duodenojejunal flexure), and place this in a purpose-built recording chamber coated with a silicone elastomer layer. 
	NOTE: The beginning of the jejunum is anatomically defined as the part of the small bowel where the ligament of Treitz crosses the small bowel, also called the duodenojejunal flexure. 
	NOTE: Cover the bottom of the recording chamber with a thin silicone elastomer layer well in advance of the experiment&#39;s start. Prepare this elastomer layer according to the manufacturer&#39;s instructions.</li>
	<li>Perfuse the chamber constantly with warm, carbogenated Krebs solution at a rate of 10 ml/min and keep the Krebs&#39; temperature in the recording chamber constant at 34 &#176;C.</li>
	<li>Mount the jejunal segment in the organ bath so that the oral end is connected to the syringe driver providing luminal flow, and the aboral end connects to the outflow. Slightly stretch the segment but take care not to exert excessive tension. Attach both ends firmly using 4/0 silk ligatures to the in- and outflow ports.</li>
	<li>Attach the syringe driver to the oral end, and perfuse the jejunal segment intraluminally with Krebs solution (non-oxygenated, ambient temperature) at a rate of 10 ml/hr.</li>
	<li>Use insect pins to pin the mesentery of the mounted intestinal segment flat against the silicone elastomer bottom layer. Stretch the mesentery out to optimize visualization of the mesenteric bundle; do not exert strain on the bundle or the jejunum.</li>
	<li>Perform a test ramp distension (vide infra) by closing the output port until the intraluminal pressure of the intestinal segment reaches 60 mmHg to verify that no intraluminal Krebs solution is leaking from the mounted segment. Observe a smooth rise in intraluminal pressure without interruptions.</li>
	<li>Observe small contractions of the segment (peristaltic waves) during the initial distension phase. If required, block peristaltic activity by adding 1 &#181;M of the L-type calcium channel blocker nifedipine to the Krebs solution. 
	NOTE: Adding 1 &#181;M of atropine to the Krebs solution in addition to nifedipine will completely paralyze the intestinal segment.</li>
	<li>Under a stereomicroscope, gently start to peel off the fat tissue from the mesentery by gently tugging it with two small tweezers, taking care not to damage the vessels and afferent nerve that lie buried in the fat tissue.</li>
	<li>Start at a remote distance from the jejunum, and expose both blood vessels in the mesenteric bundle.</li>
	<li>Observe the afferent jejunal nerve in between both vessels as a thin, white thread encapsulated in adipose tissue. Only dissect more proximally towards the jejunum by gently peeling the fat tissue away using tweezers when the initial identification of both mesenteric vessels and/or the afferent nerve is difficult.</li>
	<li>Dissect the jejunal mesenteric nerve of the segment free over a distance of several millimeters, by removing the adipose tissue adherent to the nerve.</li>
	<li>Transect the nerve using sharp tissue scissors. If required, peel off the remaining fat and connective tissue, as well as the epineuronal sheath, by gently tugging it with the small tweezers.</li>
	<li>Using a micromanipulator, lower the tip of the suction electrode, connected to a syringe with a plunger, into the organ bath; then, by manipulating the plunger, gently aspirate some Krebs solution from the organ bath so that the tip of the electrode is submerged in the Krebs solution (Figure 1). Ensure that the Krebs solution covers the wire electrode inside the suction electrode. 
	NOTE: Use a pipette puller to prepare the borosilicate glass suction capillary containing the wire electrode before the experiment starts.</li>
	<li>Position the tip of the suction electrode immediately next to the transected afferent nerve strand and draw the transected nerve strand into the capillary over its entire length.</li>
	<li>Maneuver the tip of the electrode towards some adipose tissue and aspirate this into the glass capillary while firmly aspirating with the plunger, thereby mechanically &#39;sealing&#39; the nerve in the capillary from the contents of the organ bath.</li>
	<li>Verify the recording of afferent nerve activity using the data acquisition system, e.g., by performing a ramp distension-induced increase in afferent firing (vide infra). Following the isolation of the nerve into the suction electrode, stabilize the preparation for 15 min in order to obtain a steady state of spontaneous afferent nerve activity before performing the actual experiments.</li>
	<li>To perform ramp distension, distend the intestinal segment by closing the output port, leading to a gradual rise in pressure (up to 60 mmHg). Only perform the desired experimental protocol when three consecutive ramp distensions (with a 15-minute interval) yield a reproducible multi-unit discharge (Figure 2).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>VWR Chemicals</td>
			<td>2,78,10,295</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Acros organics</td>
			<td>196770010</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate (NaH2PO4)</td>
			<td>VWR Chemicals</td>
			<td>1,06,34,61,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Merck</td>
			<td>1,06,32,91,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Merck</td>
			<td>1,05,88,61,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Merck</td>
			<td>2,38,11,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>VWR Chemicals</td>
			<td>1011175P</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td>compound Krebs solution</td>
		</tr>
		<tr>
			<td>PVC tubing</td>
			<td>Scientific Laboratory Supplies</td>
			<td></td>
			<td>The intestinal segment should be mounted over PVC tubing</td>
		</tr>
		<tr>
			<td>Silicone tubing</td>
			<td>Scientific Laboratory Supplies</td>
			<td></td>
			<td>The rest of the tubing, ideally silicone-based - more easily dislodging of debris in the tubing</td>
		</tr>
		<tr>
			<td>Silk thread</td>
			<td>Pearsall Limited</td>
			<td>10B15S220</td>
			<td>Attachment of the segment over the PVC tubing</td>
		</tr>
		<tr>
			<td>Syringe driver</td>
			<td>Harvard Apparatus</td>
			<td>55-2222</td>
			<td>Intraluminal infusion of Krebs</td>
		</tr>
		<tr>
			<td>Binocular - including 10x magnification in oculair</td>
			<td>Zeiss STEMI 2000</td>
			<td></td>
			<td>Optimal visualization for the dissection of the afferent nerve</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket Control Unit</td>
			<td>Harvard Apparatus</td>
			<td>507214</td>
			<td>Heating of the organ chamber</td>
		</tr>
		<tr>
			<td>Custom made organ bath with Sylgard covered bottom</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spike2 software</td>
			<td></td>
			<td></td>
			<td>Recording and analysis of the data</td>
		</tr>
		<tr>
			<td>Insect pins, 500 pieces, stainless steel, diameter 0.2 mm</td>
			<td>Austerlitz insect pins minutiens</td>
			<td></td>
			<td>Dissection of the afferent nerve</td>
		</tr>
		<tr>
			<td>Tweezer Dumont #5 inox 11cm</td>
			<td>World Precision Instrument</td>
			<td>500341</td>
			<td>Dissection of the afferent nerve</td>
		</tr>
		<tr>
			<td>Scissors, spring, 14 cm</td>
			<td>World Precision Instrument</td>
			<td>15905</td>
			<td>Dissection of the afferent nerve</td>
		</tr>
		<tr>
			<td>DB digitimer</td>
			<td>NL 108T2/10</td>
			<td></td>
			<td>pressure transducer</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>M-3333</td>
			<td>3D manipulation of the suction electrode</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>X-4 rotating block</td>
			<td></td>
			<td>3D manipulation of the suction electrode</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>GJ-8 magnetic stand</td>
			<td></td>
			<td>3D manipulation of the suction electrode</td>
		</tr>
		<tr>
			<td>LightSource</td>
			<td>Euromex Microscopes Holland EK-1</td>
			<td></td>
			<td>Optimal visualization for the dissection of the afferent nerve</td>
		</tr>
		<tr>
			<td>CED 1401 Recording Apparatus</td>
			<td></td>
			<td></td>
			<td>Recording of afferent nerve activity</td>
		</tr>
		<tr>
			<td>Humbug 50/60Hz Noise Eliminator</td>
			<td>Quest Scientific Instruments</td>
			<td></td>
			<td>Elimination of background noise</td>
		</tr>
		<tr>
			<td>Infusion Pump</td>
			<td>Gibson Minipuls 2</td>
			<td></td>
			<td>Infusion of the organ chamber in which the segment is mounted</td>
		</tr>
		<tr>
			<td>Microelectrode Holder Half Cells 1.5 mm</td>
			<td>World Precision Instrument</td>
			<td>MEH2SW</td>
			<td>Suction electrode for isolation of the afferent fiber</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries, 300 pc; 1.5/0.84 OD/ID</td>
			<td>World Precision Instrument</td>
			<td>1B150-4</td>
			<td>Capillary for the isolation of the afferent nerve</td>
		</tr>
	</tbody>
</table>

Source: Nullens, S., et al. <a href="https://app.jove.com/v/54576">In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments.</a> J. Vis. Exp. (2016).
This video shows the process of recording electrical signals from mesenteric afferent nerves in a dissected mouse jejunal segment to study the effects of tissue pressure and movement changes on afferent nerve activity.

<img alt="Figure 1" src="/files/ftp_upload/22842/22842_Figure1.jpg" /> 
Figure 1: Schematic Overview of the Purpose-built Recording Chamber and Suction Electrode. Detailed overview of the technical setup with the suction electrode and the recording chamber in place.
<img alt="Figure 2" src="/files/ftp_upload/22842/22842_Figure2.jpg" /> 
Figure 2: Representative Tracing of the In Vitro Recording...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Start with a recording chamber containing a dissected segment of a mouse jejunum, a part of the small intestine, submerged in an electrolyte solution and secured with a pin. 
The jejunal segment includes surrounding fat tissue with a pre-dissected mesenteric afferent nerve, which transmits signals from the jejunum to the brain. 
The segment is connected to the chamber's inflow and outflow ports for solution flow.
Take a suction electrode connected to a micromanipulator and a recording unit.
Fill the suction electrode with the electrolyte solution and position it near the mesenteric afferent nerve. 
Apply a gentle suction to pull the nerve with the surrounding fat tissue to form a tight seal.
Close the outflow port while maintaining the inflow to gradually increase the internal pressure and distension of the jejunal segment. 
This causes the excitation of the mesenteric afferent nerve around the jejunum, generating an electrical signal. 
Record the afferent nerve activity in response to jejunal pressure changes.

22 ビュー. Ex Vivoマウス空腸セグメントにおける腸間膜求心性神経活動の記録

ビデオ: Ex Vivoマウス空腸セグメントにおける腸間膜求心性神経活動の記録 - 実験

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

マウス大腸求心性終末の体外機能解析では、

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

JoVE Journal

医学

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

体外ラットの結腸求心性線維の電気生理学的特性の評価

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

神経科学

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

ゼブラフィッシュで泳ぐ後側面線の発泡性ニューロンの活動

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...